The present invention relates to a bacteria analyzer, and in particular to a time-of-flight bacteria analyzer using metastable atom bombardment ionization source.
There are presently many problems related to micro-organisms, and their rapid detection and identification is of great importance. For example, bacteria, like fungi, are involved in many human infections and it is important in clinical environments to be able to detect these organisms. In the food industry, genetically modified organisms (GMO""s) are of interest and it would be desirable to easily detect them for control purposes.
Presently, there are biological methods that can be used to identify micro-organisms but their use requires time (up to several days) which is not always desirable. For example, in clinical environments, because of the time required to get a result, physicians will often prescribe a wide-spectrum antibiotic to a patient to be on the safe side, or alternatively risk a patient""s well-being and comfort by delaying use of the correct specific antibiotic until after lab tests have identified the micro-organism source of infection. In a majority of cases, the results will come back negative and this leads to an overuse of these broad-spectrum drugs. As a consequence, the price of health care is higher, and this practice is also in part responsible for the Methods usually available for the identification of micro-organisms are based on biological processes (genotyping) and rely on amplification methods (PCR, culture, etc.). The amplification step is often the time limiting factor in obtaining a result.
Detection and identification of micro-organisms by physical processes can be done rapidly and several approaches have been described (Goodfellow M., Freeman R. and Sisson P. R., Zbl. Bakt. (1997) 285, 133-156). These approaches generally make use of analytical techniques such as gas-chromatography (GC) or mass spectrometry (MS). They usually involve a thermal process such as rapid heating of the sample to a high temperature (pyrolysis) (Fox A. and Morgan S. L., In: Rapid Detection, and Identification of Microorganisms (Nelson, W. H., ed.) pp 135-164. Vch Publishing, Deerfield, Fla., USA, 1985; Smith C. S., Morgan S. L., Parks C. D., and Pritchard D. G., Anal. Chem., (1987) 59, 1410-1413; Euly, L. W., Walla M. D., Hudson J. R., Morgan S. L., and Fox A., J. Anal. Appl. Pyrol. (1985) 7, 231-247; Walla M. D., Morgan P. Y., Fox A., and Brown A., J. Chromatog. (1984) 288, 399-413; Reiner E., and Ewing W. H., Nature (1968) 217, 191-194; Gutteridge C. S. and Norris, J. R., Appl. Environ. Microbiol., (1980) 40, 462-465; Soderstrom B., Wold S. and Blomquist G., J. Gen. Microbiol., (1982) 128, 1773-1784; Bayer F. L., and Morgan S. L., In: Pyrolysis and GC in Polymer Analysis (Liebman S. A. and Levy E. J., eds) pp. 277-337, Marcel Dekker, N.Y., USA, 1985; Meuzelaar H. L. C., Haverkamp J. and Hileman F. D., Pyrolysis Mass Spectrometry of Recent and Fossil Biomaterials, Elsevier, Amsterdam, 1982; Irwin W. J., J. Anal. Appl. Pyrol., (1979) 1, 89-122) or exposition of the sample to a laser beam (Hiilemkamp F., Karas M., Beavis R. C. and Chait B. T., Anal. Chem. (1991) 63, 1193A-1202A), e.g. matrix-assisted laser desorption/ionization (MALDI). In the pyrolysis approach, the micro-organism is rapidly heated, in the absence of oxygen, to a high temperature which leads to the thermal breakdown of the sample, thus generating secondary products that can be used as markers for identification of the micro-organisms. The decomposition products can be analyzed by gas-chromatography (as methyl esters of fatty acids) (Py-GC) or by mass spectrometry (Py-MS). When the sample is exposed to a laser beam (MALDI), the micro-organisms are deposited on a probe, under vacuum, and bombarded by a laser beam pulse of high energy. In Py-MS and MALDI, a mass spectrometer is used to analyze the decomposition products by monitoring mass spectra during the decomposition process. Both approaches have limitations, since in Py-MS techniques, variability due to the ionization technique can cause problems (generation of exportable libraries of micro-organism fingerprints) and in MALDI, the micro-organism has to be inserted into a solid matrix which reduces the universality of the process (different matrices have to be used for different micro-organisms) and reduces the detection limits.
Although Py-MS techniques have a potential to provide rapid answers to micro-organism detection and identification, they have been limited because of problems generated mainly by the ionization technique used in Py-MS. These problems stem from the fact that, in many cases, pyrolysis has to be conducted away from the ionization chamber and that the ionization process itself is not adequate leading to a loss of information and a complication of the mass spectra obtained during pyrolysis.
In many instances, pyrolysis of the sample (micro-organism or polymer) is conducted in a chamber remote from the ionization source and the decomposition products are carried to the ion source of the mass spectrometer by an inert carrier gas (usually Argon) through a capillary. The resulting effects of this approach are that compounds (radicals or molecules) issued from the primary process of pyrolysis are lost. For example, high molecular weight species that have a low vapor pressure can condense on the walls of the capillary and reactive species (radicals) can react at the walls or be recombined. In both of these cases, high molecular weight species are not monitored by the mass spectrometer and because they contain a high degree of information, specificity is lost.
The ionization process used in the mass spectrometer can play a key role in the detection and identification of the micro-organism. In early studies, electron ionization was used to ionize products generated during pyrolysis. This ionization technique leads to complex mass spectra containing mostly low molecular weight ions. The complexity of the mass spectra is due to the fact that electron ionization is a very energetic process that induces extensive fragmentation. Thus, fragments generated during pyrolysis are refragmented in the ion source of the mass spectrometer yielding a legion of ions most of which are at low masses. Because of this extensive fragmentation, high molecular weight species that contain specific information on the identity of the compound are destroyed and the information is lost. Attempts have been made to remedy this problem. An approach is to lower the electron energy, thus, reducing fragmentation upon ionization. However, lowering the electron energy significantly reduces sensitivity (by more than one hundred) and leads to irreproducible results because of the overwhelming effect of source tuning conditions at low energy. Hence, it becomes almost impossible to generate libraries of spectra of micro-organisms that can be exported to other laboratories.
Recent studies have been conducted to improve the Py-MS approach. In these studies, methylation is conducted during pyrolysis and ionization is achieved with chemical ionization. The methylation step aims at increasing the volatility of the compounds formed during pyrolysis, thus, increasing their chance of reaching the ion source and being ionized. Combined with the methylation step, chemical ionization (Barshick S. A., Wolf D. A. and Vass A. A., Anal. Chem. (1999) 71, 633-641) is used to reduce the limitations found in electron ionization. Because chemical ionization is a softer method than electron ionization, in theory, it should favor the presence of higher mass ions. In practice, chemical ionization combined with methylation yields higher mass fragments (up to m/z 300) but because of the presence of a high pressure plasma in the ion source, that is necessary for the chemical ionization process, other complications are found. The presence of a reagent gas at high pressure creates a high background signal, thus, creating interferences and reducing the sensitivity of the approach.
According to a first object of the invention, micro-organism or other very high molecular weight micro-objects are analyzed using a physical process rather than a biological process. Thus, an instrument (Bacteria analyzer) has been developed and is provided which allows a fingerprint of micro-organisms to be obtained rapidly (within minutes), thus providing a means for their rapid detection and identification.
According to a broad aspect of the invention, an analyzer for bacteria or other micro-organism-like micro-objects has been developed which uses an xe2x80x9cin-beamxe2x80x9d pyrolyzer, a metastable atom bombardment ionization source, and a time-of-flight (TOF) mass analyzer to conduct rapid detection and identification of micro-organisms and chemical polymers.
The approach that is described in this application can remedy both types of problems associated with pyrolysis having to be conducted away from the ionization chamber and the ionization process itself being inadequate leading to a loss of information and a complication of the mass spectra obtained during pyrolysis.
The present invention, uses xe2x80x9cin-beamxe2x80x9d pyrolysis where the sample is pyrolyzed directly in the source (xe2x80x9cin-beamxe2x80x9d) of the mass spectrometer therefore providing high-mass information from the compound being analyzed. Ions at high mass are much more specific in terms of biomarkers and therefore provide specific information on the identity of the system being under investigation.
The present invention remedies most of the problems described previously by reducing fragmentation, increasing sensitivity and reproducibility and provides means by which high mass markers can be monitored. It also allows fingerprint of the micro-organisms or chemical polymers to be obtained at several precisely known ionization energies which increases the selectivity of the technique. The increase in reproducibility due to the use of quantized energies for ionization allows spectral libraries of micro-organisms to be generated and these are exportable to other laboratories because the excitation energy of the metastable species is not affected by experimental conditions.
According to a first independent aspect of the invention, an analyzer apparatus for high molecular weight species is provided. The apparatus comprises a metastable atom generator, and a pyrolyzer for pyrolysis of a sample of the high molecular weight species, an ionization chamber in communication with the generator and the pyrolyzer. The ionized ones of said species are accelerated by an electric extraction lens device into a mass analyzer.
Preferably, the mass analyzer is a time-of-flight (TOF) analyzer. The metastable atom generator preferably outputs a beam of metastable atoms along an axis extending through the chamber and the lens device into the mass analyzer. The chamber may comprise a conical repeller-deflector having an orifice at its apex through which the metastable atoms pass.
The ion chamber preferably comprises a slit through which pyrolyzed product passes from the pyrolyzer in a direction perpendicular to the beam axis. The beam of metastable atoms is preferably substantially free of ions.
According to a second independent aspect of the invention, there is provided a method of analyzing a micro-organism comprising the steps of preparing a sample of the micro-organism, placing the sample in a pyrolyzer, pyrolyzing the sample with a selected temperature program to provide pyrolyzed product of a high-dalton mass range, ionizing the product using metastable atoms, and analyzing the ionized product using a high acquisition rate mass analyzer. It is preferred that the product is provided directly in an ionization chamber, and that the metastable atoms are provided by a beam traversing the chamber and passing into the analyzer.